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Abstract

The proliferative rate of chondrocytes affects bone elongation. Chondrocyte hypertrophy is required for endochondral bone formation as chondrocytes secrete factors required for osteoblast differentiation and maturation. Previous studies have demonstrated that the Indian hedgehog (Ihh) signaling pathway is a key regulator of skeletal development and homeostasis. The aim of the present study was to investigate the function of Ihh in chondrocyte proliferation and differentiation, as well as the underlying mechanisms. Ihh was knocked down in mouse chondrocyte cells using short hairpin RNA. Chondrocyte apoptosis and cell cycle arrest were assessed using flow cytometry and the results indicated that knockdown of Ihh significantly inhibited cell growth (P<0.05) and increased apoptosis (P<0.001) compared with negative control cells. Downregulation of Ihh also resulted in cell cycle arrest at G1 to S phase in chondrocytes. It was also observed that knockdown of Ihh decreased alkaline phosphatase activity and mineral deposition of chondrocytes. The inhibitory roles of Ihh downregulation on chondrocyte growth and differentiation may be associated with the transforming growth factor‑β/mothers against decapentaplegic and osteoprotegerin/receptor activator of nuclear factor κB ligand signaling pathway. The results of the present study suggest that chondrocyte‑derived Ihh is essential for maintaining bone growth plates and that manipulation of Ihh expression or its signaling components may be a novel therapeutic technique for the treatment of skeletal diseases, including achondroplasia.

Introduction

Bone formation in the developing embryo is governed
by intramembranous and endochondral ossification initiating from
mesenchymal condensations, in which mesenchymal progenitor cells
differentiate (1). During
endochondral ossification, mesenchymal progenitors differentiate
into chondrocytes to form the cartilage template of the bone, which
is required for bone elongation. Chondrocyte hypertrophy is
required for endochondral bone formation; elongated chondrocytes
exit the cell cycle to become prehypertrophic chondrocytes prior to
terminally differentiating to form hypertrophic chondrocytes
(2). These chondrocytes subsequently
secrete factors required for osteoblast differentiation and
maturation (3).

It has previously been reported that the Indian
hedgehog (Ihh) signaling pathway is a key regulator of skeletal
development and homeostasis (4) and
that Ihh signaling must be enhanced to increase bone formation
(5). In a previous study,
overexpression of Ihh ameliorated short stature homeobox 2 (shox2)
overexpression-associated reduction of extracellular matrix
components; however, it did not inhibit the increase in matrix
metalloproteinase (MMP)9 and MMP13, or apoptosis in the
temporomandibular joint, which suggests that overexpression of Ihh
only partially inhibits shox2 overexpression-associated congenital
dysplasia of the temporomandibular joint (6). Dysregulation of Ihh signaling results
in multiple bone diseases, including progressive osseous
heteroplasia (7). Treatment with an
Ihh signaling inhibitor has been reported to reduce the occurrence
of chondroma-like lesions, including enchondromas and
osteochondromas, adjacent to disordered growth plates in fibroblast
growth factor receptor 3-deficient mice (8). Ihh deletion also induces symphalangism,
characterized by initial cartilaginous fusion preventing epiphyseal
growth plate formation, resulting in abnormal directionality of
chondrocyte differentiation in mutant mice (9). Ihh signaling serves an important role
in the mineralization process of fibrocartilaginous entheses
(10). Inhibiting Hh signaling
reduces mineralized fibrocartilage, leading to less collagen
embedded within mineralized tissue (11). Understanding the signaling mechanisms
and functions of Ihh signaling in bone development may provide
important insights into bone disease prevention and
therapeutics.

In the present study, Ihh was knocked down in
chondrocytes using short hairpin (sh)RNA to investigate the
function of Ihh signaling in chondrocyte proliferation and
differentiation. The present study also aimed to explore the
potential mechanism by which Ihh induces chondrocyte apoptosis and
cell cycle arrest.

Cell Counting Kit-8 (CCK-8) cell
proliferation assay

Cell proliferation rates were measured using CCK-8
(Beyotime Institute of Biotechnology, Haimen, China). A total of
0.5×104 cells were seeded in 96-well plates for 24 h,
transfected with the indicated lentivirus, and further incubated
for 24, 48 and 72 h at 37°C, respectively. A total of 10 µl CCK-8
reagents were added to each well at 1 h prior to the endpoint of
incubation (23, 47 and 71 h). The optical density of each well was
subsequently determined using a microplate reader at a wavelength
of 490 nm.

An Annexin V Apoptosis Detection kit (Thermo Fisher
Scientific, Inc.) was used to assess apoptosis. Following
incubation for 24, 48 or 72 h at 37°C, the cells were trypsinized
for 2 min at 37°C, collected by centrifugation (960 × g) for 5 min
at room temperature and resuspended in 2 ml medium. Approximately
2×105 cells were harvested, washed twice with cold PBS
and resuspended in 500 µl binding buffer provided in the kit. A
total of 10 µl Annexin V-fluorescein isothiocyanate and 10 µl PI
was added to the solution and mixed well. Following incubation for
15 min at 37°C, cells were analyzed using a flow cytometer (BD
Biosciences, San Jose, CA, USA).

Cell cycle analysis assay

Cell cycle analysis was determined by flow cytometry
using a Cell Cycle and Apoptosis Analysis kit (cat no. C1052,
Beyotime Institute of Biotechnology) and analyzed the data using
Flowjo (7.6 version; Flowjo LLC, Ashland, OR, USA). Briefly,
~1×104 cells were seeded in each well of a 6-well plate.
Following 48 h transfection with Ihh-shRNA or empty lentivirus,
cells were harvested and fixed in 70% ice-cold ethanol for 24 h,
followed by staining with PI for 10 min at room temperature. The
different phases of the cell cycle were analyzed using a
FACSCalibur flow cytometer (BD Biosciences).

Determination of mineralization using
von Kossa staining

Von Kossa staining (Sigma-Aldrich; Merck KGaA,
Darmstadt, Germany) was used to determine the extent of minerals
deposited on chondrocyte cells. Silver nitrate solution was added
to the cells and the plate was exposed to ultraviolet light for 30
min. Cells were subsequently washed with PBS and the reaction was
stopped with the addition of 500 µl of 5% sodium thiosulfate
(Sigma-Aldrich; Merck KGaA). The positive mineral depositions were
stained in black. Cells were counterstained with nuclear fast red
for 5 min at room temperature to label the nucleus and cytoplasm.
Images were captured using an inverted microscope (magnification,
×100; Nikon Eclipse TC 100; Nikon Corporation).

Statistical analysis

All data are expressed as the mean ± standard
deviation of three independent experiments. Statistical analyses
were performed using SPSS v. 17.0 (SPSS, Inc., Chicago, IL, USA).
Differences between groups were analyzed using Student's t-test or
one-way analysis of variance with Tukey's post hoc test dependent
on the conditions. P<0.05 was considered to indicate a
statistically significant difference.

Results

To investigate the biological role of Ihh in
chondrocyte cell growth, chondrocytes transfected with Ihh shRNA
were used in the present study. The results of RT-qPCR revealed
that Ihh expression was significantly reduced in transfected cells
compared with NC cells (P<0.001; Fig.
1A). Ihh downregulation significantly inhibited cell growth
(P<0.05; Fig. 1B) and
significantly increased the apoptosis rate compared with the NC
group (P<0.001; Fig. 2A). In
addition, Ihh knockdown significantly reduced the percentage of
cells in S phase (P<0.05) and increased the percentage of cells
in G1 phase compared with the NC group (P<0.01), indicating cell
cycle arrest at G1 to S phase (Fig.
2B).

Effects of Ihh on chondrocyte cell
differentiation

ALP is an enzyme that is required for bone
mineralization (13). ALP activity
was assessed in the present study and was markedly decreased in Ihh
knockdown cells compared with the NC group (Fig. 3A). The calcification and
mineralization of bone matrix are essential for the strength and
rigidity of the spinal skeletal system (14). To estimate mineralization and
calcification, von Kossa staining was performed following Ihh
knockdown. Representative images of von Kossa staining were
obtained by bright field microscopy (Fig. 3B). Compared with the NC cells,
mineral deposition was markedly decreased in the Ihh-shRNA
group.

Effects of Ihh on TGF-β/Smads and
OPG/RANKL signaling

The effects of Ihh on TGF-β/Smads and OPG/RANKL
signaling were also investigated. The mRNA expression of COL10A,
RANKL, Samd2 and Smad3 was significantly upregulated in the
Ihh-shRNA group compared with the NC cells (P<0.001; Fig. 4), whereas the levels of OCN, OPG,
PTHrP, Runx2 and TGF-β were significantly downregulated in the
Ihh-shRNA compared with the NC group (P<0.001; Fig. 4). Protein expression of these genes
was further assessed by western blot analysis (Fig. 5). The protein expression of COL10A,
RANKL, Samd2 and Smad3 were upregulated, whereas levels of OCN,
OPG, PTHrP, Runx2 and TGF-β were significantly downregulated in Ihh
knockdown cells compared with the NC group (P<0.05; Fig. 5). These findings suggest that the
inhibitory effects of Ihh knockdown on bone differentiation and
generation are achieved via the TGF-β/Smads and OPG/RANKL signaling
pathway.

Discussion

In the present study, Ihh knockdown was demonstrated
to significantly inhibit cell growth and increase the apoptosis
rate compared with NC cells. Downregulation of Ihh resulted in cell
cycle arrest at G1 to S phase in chondrocytes. In addition, Ihh
knockdown decreased the ALP activity and mineral deposition of
chondrocytes. These results suggest that the inhibitory roles of
Ihh downregulation on chondrocyte growth and differentiation may be
associated with the TGF-β/Smads and OPG/RANKL signaling
pathways.

Ihh signaling is essential for chondrocyte
differentiation and endochondral ossification (15). Deletion of Ihh in postnatal
chondrocytes in a temporal/spatial-specific manner results in the
loss of ectopic hypertrophic chondrocyte formation in the growth
plate (16). In a previous study,
mutant mice with Ihh deletion exhibited a continuous loss of
trabecular bone over time, which indicates that postnatal
chondrocyte-derived Ihh is essential for skeletal growth,
maintaining the growth plate and sustaining trabecular bone growth
(16). A previous report
demonstrated that treating chondrogenic cells with an Ihh inhibitor
inhibited chondrocyte differentiation and secondary ossification
center formation (17). Furthermore,
Gualeni et al (18)
previously demonstrated that diastrophic dysplasia mouse
proteoglycan undersulfation resulted in reduced chondrocyte
proliferation in the proliferative zone via the Ihh pathway,
contributing to reduced long bone growth. Ihh expression has been
reported to be significantly increased in humans with the modic
degeneration I and II groups and is positively correlated with the
severity of degeneration (19).
Overexpression of Ihh signaling promotes abnormal chondrocyte
differentiation in endochondral ossification and enhances bone
formation in posterior longitudinal ligaments (20). Ihh is synthesized by chondrocytes and
is required for the synthesis of PTHrP (21). Ihh acts with PTHrP in a negative
feedback loop to regulate early chondrocyte differentiation and
hypertrophic differentiation (22).
Blocking Ihh signaling with cyclopamine has been reported to delay
chondrocyte hypertrophy in PTHrP knockout embryos, whereas
upregulating Ihh signaling in the postnatal cartilage led to
accelerated chondrocyte hypertrophy during secondary ossification,
indicating that Ihh signaling promotes chondrocyte hypertrophy
independently of PTHrP, which may be mediated by Wnt/β-catenin
signaling (23,24). As chondrocytes go through a program
of proliferation and subsequent differentiation into hypertrophic
chondrocytes, PTHrP maintains chondrocyte proliferation and delays
their further differentiation (25).
The differentiation-delaying action of PTHrP is mediated by
suppressing the synthesis of Runx2, which is a transcription factor
integral to osteoblast differentiation (26). In the absence of Ihh, the osteoblast
fails to activate the expression of Runx2 (27). However, forced expression of Runx2 in
the skeletogenic cells restores bone formation in the Runx2-null
embryo, whereas it does not in the Ihh-null embryo; this suggests
that Ihh-induced osteoblast differentiation requires additional
effectors (27).

The results of the present study demonstrate that
the mRNA and protein expression of hypertrophic markers, including
COL10A, RANKL, Smad2 and Smad3, are upregulated in chondrocytes
following Ihh knockdown, whereas levels of OCN, OPG, PTHrP Runx2
and TGF-β are significantly reduced. Previous studies have reported
that multiple signaling pathways, including Wnt/β-catenin and
TGF-β/Smad pathways, are able to regulate chondrocyte hypertrophy
(28). The Smad2/3 pathway is
directly activated by TGF-β, which leads to inhibited hypertrophy
(29). TGF-β activation of Smad3
also inhibits Runx2 via epigenetic regulation (30). Glioma-associated oncogene-Krüppel
family members (Gli) 1 and 2, which are Ihh downstream
transcription factors, increase COL10A activity and Runx2 promotes
COL10A1 expression via interacting with Ihh (31). Furthermore, Gli1 and Gli2 act in a
complex with Runx2/Smad to induce chondrocyte differentiation
(30), suggesting that Ihh signaling
may be an important factor for early chondrocyte differentiation
and the maturation and calcification of chondrocytes.

In conclusion, knockdown of Ihh suppresses
chondrocyte growth and differentiation and this effect may be
associated with the TGF-β/Smad and OPG/RANKL signaling pathways.
These results suggest that chondrocyte-derived Ihh is essential for
maintaining the growth plate and that manipulating Ihh expression
or its signaling components may be a novel effective treatment for
achondroplasia and other skeletal diseases.

Acknowledgements

The present study was supported by the Natural
Science Foundation of Hunan Province (grant no. 2016JJ3160) and the
National Natural Science Foundation of China (grant no.
81472145).